Epidemiological and genetic studies have implicated decreased plasma levels of high density lipoproteins (HDL) in the pathogenesis of atherosclerosis, whereas increased HDL levels have been correlated with protection against coronary heart disease and associated with longevity (Karathanasis, S., et al., Proc. Natl. Acad. Sci. USA 80: 6147-6151 (1983)). The molecular bases for these observations have not been elucidated, but it has been shown experimentally that HDL stimulates cholesterol efflux from cells and may in this manner act as a carrier for reverse cholesterol transport, i.e., the delivery of cholesterol from the peripheral tissues to the liver for disposal (ibid.). This mechanism could in part explain the inverse correlation observed between the size of total body cholesterol pools and plasma HDL levels, and between HDL levels and coronary heart disease. It also is possible that HDL might simply be a marker of a certain pattern of lipid transport and metabolism which confers protection against the development of atherosclerosis.
Apolipoprotein A1 (apoA1) is the major protein constituent of HDL. It is thought that, because of their involvement in cholesterol excretion, apoA1 and HDL are important in protection against coronary heart disease. Indeed, genetic deficiencies in apoA1 and HDL are associated with intracellular cholesterol accumulation and premature atherosclerosis.
ApoA1 is a relatively abundant (.about.1 to 1.5 mg/ml) plasma protein of known primary structure (see Karathanasis, et al., cited above, Cheung, P., and Chan, L., Nucl. Acids Res. 11: 3703-3715 (1983), and the references cited therein). ApoA1 has been shown to be a necessary component in mixtures with phospholipid for the successful removal of cholesterol from ascite cell membranes, and the protein is a potent activator/cofactor of lecithin:cholesterol acyltransferase, a plasma enzyme that catalyzes the conversion of cholesterol and phosphatidylcholine to cholesterol esters and lysophosphatidylcholine (ibid.).
ApoA1 appears to be synthesized predominantly in the liver and small intestine of mammals, but has been found in a variety of other tissues in the rooster (ibid.). The cDNA for human hepatic apoA1 has been cloned by different laboratories (Karathanasis, et al., and Cheung and Chan, cited above). The primary translation product of apoA1 mRNA is a preproprotein that undergoes intra- and extracellular proteolytic processing to produce the major apoA1 isoprotein form observed in plasma. Deficiencies of apoA1 are associated with abnormalities in lipoprotein metabolism that result in low plasma HDL levels and may contribute to the development of premature atherosclerosis.
In addition to its imperfectly understood role in cholesterol transport and atherosclerosis, apoA1 appears to play a role in restenosis and septic shock, conditions involving circulatory disturbances. Restenosis is the narrowing or stricture of blood vessels, typically after angioplasty or surgical correction of the condition. Septic shock, associated with both gram-negative and gram-positive infections, is characterized by inadequate vasomotor tone resulting in profound hemodynamic disturbances involving a marked decrease in peripheral vascular resistance. Recently, purified apoA1 has been shown to prevent endotoxin-induced monokine release by human low- and high-density lipoproteins (Flegel, W. A., et al., Infection and Immunity 61: 5140-5146 (1993)).
Studies aimed at establishing the mode of action of apoA1, including its role in pathological processes and in lipid metabolism, and elucidation of its crystal structure, have been hampered by the lack of sufficiently large amounts of the protein. Isolations from plasma are arduous. The protein has been expressed in Chinese hamster ovary cells, but only about 30% yields of secreted protein have been achieved (Mallory, J. B., et al., J. Biol. Chem. 262: 4241-4247 (1987)). Attempts have been made to express the protein in bacteria as a .beta.-galactosidase fusion protein, but the apoA-1 fusion product was found to be sensitive to degradation in culture (ibid.) The protein was expressed in E. coli, but the heterologous protein was unstable with a half-life of &lt;10 minutes as determined by pulse-chase experiments (ibid.).
It would be desirable to have large quantities of apoA1 available at a reasonble cost.